Targeted DNA insertion in tomato using RNA-guided nucleases

Grandgeorge, Lila (2021) Targeted DNA insertion in tomato using RNA-guided nucleases. Doctoral thesis, University of East Anglia.

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Insertion of novel DNA sequences at defined locations in plant genomes, known as a knock-in (KI), is highly desirable due to its potential for crop trait improvement. However, KIs are difficult to achieve. Previous KI attempts showed variable efficiencies (0.1% to 25%) in different plant species. I aimed to establish a high efficiency KI protocol in tomato and tested a range of variables for their ability to improve rates of KIs by homologous recombination. A 35S promoter or 35S enhancer was targeted upstream of the tomato ANT1 gene, leading to purple pigmentation of tissues upon successful insertion and this was scored to measure KI efficiency. Variables tested included induction of double-stranded breaks (DSB) at the genomic target using CRISPR/Cas9 or a nickase allele to deliver single stranded breaks. Two viral replicons based on different strains (acute or mild) of a Bean Yellow Dwarf virus (BeYDV) Geminivirus were tested to provide a high copy number of the donor template, and for assessing replicon cargo size impact on KI efficiency. In these experiments, KI efficiencies were low and did not reach above 3%. Regeneration of edited plants from excised purple sectors was challenging due to overgrowth of surrounding wild-type cells. To limit the growth of “escape” background tissues and improve the regeneration of plant material containing a KI, I tested the use of a temperature-dependent selectable marker (Degron-NptII) to eliminate cells enabled to survive on selective medium through transient NptII expression rather than stable insertion of the T-DNA. Experiments demonstrated the degron-NptII strategy improved the selection precision of transgenic tissues at the callus, shoot and root formation stages and reduced the occurrence of escapes. Incorporating the degron-NptII strategy and removing the viral replicon (suspected to interfere with the regeneration process) from transformation vectors, further variables were tested for high efficiency KI. Compared to Cas9, a temperature tolerant LbCas12a (ttCas12a) allele achieved higher KI rates (26.2% compared to 20.7%). Inducing three DSBs (one DSB at target site and two at extremities of the donor fragment on T-DNA) instead of one DSB (at genomic target) increased rates of KIs when using Cas9 (12% compared to 22%). The three DSBs approach improved rates of KIs to a lesser extent compared to one DSB when utilising ttLbCas12a (24.2% compared to 28.2%). Additionally, the blight resistance gene Rpi-vnt1 was knocked in alongside a 35S promoter, making a 7.3 kb DNA insert and resulted in a mean of 27.8% KI efficiency. True KI events with full R gene insertion were confirmed by PCR and Sanger sequencing in several samples.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 11 May 2022 13:35
Last Modified: 11 May 2022 13:35

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